Exploring the therapeutic potential of sodium deoxycholate tailored deformable-emulsomes of etodolac for effective management of arthritis

The current piece of research intends to evaluate the potential of combining etodolac with deformable-emulsomes, a flexible vesicular system, as a promising strategy for the topical therapy of arthritis. The developed carrier system featured nanometric dimensions (102 nm), an improved zeta potential (− 5.05 mV), sustained drug release (31.33%), and enhanced drug deposition (33.13%) of DE-gel vis-à-vis conventional system (10.34% and 14.71%). The amount of permeation of the developed nano formulation across skin layers was demonstrated through CLSM and dermatokinetics studies. The safety profile of deformable-emulsomes has been investigated through in vitro HaCaT cell culture studies and skin compliance studies. The efficacy of the DE-gel formulation was sevenfold higher in case of Xylene induced ear edema model and 2.2-folds in CFA induced arthritis model than that of group treated with conventional gel (p < 0.01). The main technological rationale lies in the use of phospholipid and sodium deoxycholate-based nanoscale flexible lipoidal vesicles, which effectively encapsulate drug molecules within their interiors. This encapsulation enhances the molecular interactions and facilitates the transportation of the drug molecule effectively to the target-site. Hence, these findings offer robust scientific evidence to support additional investigation into the potential utility of flexible vesicular systems as a promising drug delivery alternative for molecules of this nature.


Micromeritics studies
The three most important parameters of micromeritics i.e., size of the vesicles, polydispersity index and zeta potential were measured with laser diffraction (measure hydrodynamic diameter) method, using Malvern Nanoseries manufactured by Malvern Instruments Ltd, UK.An aliquot of 1 mL of DE suspension was diluted 100 times with distilled water followed by the measurement of vesicle size and PDI.The sample was bath sonicated for 1 min using a bath sonicator at 25 °C.The zeta potential was measured of the undiluted sample at 25 °C (23.2 V/cm electric filed strength) and reported as an average value of three measurements 36 .

Drug entrapment efficiency and drug content
The drug entrapment was determined using centrifugation technique (Remi, RM-12C, Mumbai, India).A 2 mL microcentrifuge tube containing ETO loaded DE vesicular suspension was subjected to centrifugation at a speed of 20,000 rpm (266,866 g) for approximately 0.5 h.The clear supernatant obtained was subsequently subjected to analysis for the presence of unentrapped drug using HPLC.The quantification of the entrapped drug was achieved by subtracting the amount of unentrapped drug from the total quantity of drug that was initially added.The same procedure was carried out for emulsomes prepared without any drug (blank) to rule out interference by any other excipients.Percent drug entrapment (PDE) determined as described in Eq. (1) 37 .Similarly, the % drug content from DE-gel was determined using sonication assisted extraction with acetonitrile.Accurately weighed (proximately 0.5 gm) of DE gel was placed in a 100 mL volumetric flask.Approximately 30 mL of acetonitrile was introduced into the flask, followed by sonication and agitation for a duration of 30 min.The sonicated solution should be cooled and subsequently diluted up to the mark with acetonitrile.The sample was filtered through a 0.45 m membrane filter and analyzed using HPLC after suitable dilutions.The % drug content calculated using Eq.(2) 26 .
(1) % Drug Entrapment = Entrapped drug in (mg) Total drug added (mg) × 100 www.nature.com/scientificreports/Vesicle count A 10-time dilution of ETO loaded DE was prepared in distilled water and kept on Haemocytometer grid (Neubauer, Fein-Optik, Germany).The vesicle density per volume was determined using microscope.The number of vesicles in 144 small squares were counted and Eq. ( 3) was further used to calculate the number density 35 .

Deformability index
Deformability index of ETO-loaded DE was measured employing vesicle-extruder (Eastern Sci.Inc., USA).A vesicle extruder was used to pass the vesicular suspension through polycarbonate membrane filter with a size of 50 nm.The size of vesicles was measured initially and then after the extrusion process using Malvern Zeta Sizer Nanoseries.The deformability index was calculated using Eq. ( 4) 29,35 .
PE is particle size measured before any extrusion (n = 3) and PA is average particle size measured after the process of extrusion (n = 3).

Morphological evaluation
The topographical and morphological traits of DE were assessed using Field Emission-Scanning Electron Microscope (FESEM; Hitachi SU8010, Japan).A drop of DE suspension was placed on a carbon coated copper grid for 15-30 min for drying which were further coated with gold.All samples were examined at an accelerated voltage of 5.0 kV and magnification of 220×and 20,000× 38,39 .

Calorimetric studies
Differential scanning calorimetry (DSC) curves of ETO, PL90G, Cap MCM C10, Cr A25), SDC and DE formulation were noted using DSC Q20 TA (M/s PerkinElmer Inc. USA). 5 mg of the sample was weighed into a hermetically sealed pan made of aluminium and further heated at 10 °C/min over 20˚C and 280˚C temperature range.The flow of nitrogen was maintained at 22 mL/min.Each sample was measured for three runs.The DSC curves were further studied using DSC software(s) (i.e., Star e and Universal) and scans were recorded 40 .

Infra-red spectroscopy
Infrared spectra of ETO, PL90G, Cap MCM C10, ETH, Cr A25), SDC and DE formulation were determined employing (Spectrum Two™ M/s PerkinElmer Inc., USA) at temperature of 25 °C and wave number 3500 cm −1 -500 cm −1 .Potassium bromide (KBr) was mixed with the sample in the ratio of 2:98 to prepare a pellet 41 .FTIR measurements were carried out using potassium bromide (KBr) technique where small amount of the each samples were dispersed with KBr to form a pellet followed by spectra acquisition in transmission mode 42,43 .

Evaluation of ETO loaded-DE gel formulation pH measurement
pH of prepared DE-gel was investigated using pH meter (Cyber Scan, Eutech Instruments Pte Ltd., Singapore).The assessments were carried out three times and the average numbers were premeditated 26 .

Rheological studies
The rheological study of DE-gel formulation was done using cone and plate type rheometer (Rheolab QC, Anton Paar GmbH, Vienna, Austria).The rheology measurement was carried out at a varying shear rate increased from 0 to 100 s −1 at temperature of 30 °C44 .The relationship between shear stress (τ) and shear rate (γ) was analysed using Herschel-Bulkey Model as represented in Eq. ( 5) 45 .
In the given equation, k represents the consistency index, τ 0 denotes the yield stress, and n signifies the power law-exponent.

Texture analysis
To determine other rheological features, i.e., consistency, firmness, cohesiveness (stickiness) and viscosity index of the formulations texture profile analysis of DE gel was performed by employing TTC spreadability rig fitted on Texture Analyzer™ (M/s Stable Micro Systems Ltd., UK) 42,46 .
(3) No. of vesicles per cubic mm = No. of vesicles in small squares x Dilution factor × 4000 Total no. of small squares counted .

Ex vivo permeation studies on mouse skin
The permeation experiments were performed employing using a diffusion assembly prepared in-house.The apparatus is composed of two reservoirs including donor and receiver compartment along with the port where sampling is done.The skin tissue was clamped on the hollow tube end exposed to the receptor phase lying underneath.The skin from a single rat was used for six diffusion cell assembles to avoid variations in the data.
Weighed quantity (~ 0.5 g) of DE-gel and conventional gel, containing ETO equivalent to 2% w/w were introduced uniformly into the donor compartment.The cross-sectional mean surface area available for diffusion was 3.15 cm 2 and receptor volume was 40 mL.The receiver media with a composition of phosphate buffer saline (pH 7.4) and ETH (80:20, %v/v) was stirred at 200 rpm.ETH was incorporated in the diffusion medium to maintain the sink condition 30 .The temperature of the receptor medium was maintained at 32 ± 0.5 °C by warm water in the outer jacket (i.e., glass beaker) of the cells employing a thermostatically controlled magnetic stirrer to equilibrate the system. 1 mL volume of samples were removed at suitable intervals for a period of 24 h and replaced with fresh media to maintain the receptor volume.The collected samples were subsequently diluted and subjected to quantitative analysis utilizing HPLC 47 .

Drug deposition studies in skin
After 24 h of skin permeation studies, the mounted tissue was carefully separated from the diffusion cell assembly.
Then skin tissue was washed three times with triple-distilled water and further dried with cotton swab.Later, the tissue was then cut in to small fragments and mixed with 5 mL ACN and shaken for 12 h at 32 ± 1 °C to completely extract ETO.After 12 h supernatant was removed and filtered and subjected to drug content analysis (in triplicate) by using HPLC 48 .

Skin depth profiling using confocal microscopy
The dye loaded formulation was prepared by method as adopted in Sect."Fabrication of ETO loaded deformable-emulsomes" except drug is replaced with coumarin-6 at a concentration of 0.15 µmol/mL.Dye loaded DE formulation and coumarin-6 dye alone were applied for 6 h to the rat skin followed by rinsing the skin with phosphate buffer (pH 7.0) and the fixed slides were prepared.Fluorescence in different skin layers was visualized with Laser Scanning Microscopy (CLSM; Nikon Eclipse Ti M/s Nikon Instruments Inc., Melville, USA) 42,49 .

Dermal kinetic studies
Dermatokinetic analysis of developed DE-gel formulations was carried out on excised skin tissue from Wistar rats in comparison with conventional gel.The tissue was mounted on diffusion cell assembly (in house assembly).Weighed quantity (0.5 g) of DE-gel, was introduced uniformly into the donor chamber.The receptor compartment had similar composition as previously explained in ex vivo permeation studies.The assembly was continuously stirred for 12 h so that sink conditions are maintained.The mounted skin was collected from each diffusion cell at subsequent time intervals and removed skin was washed three times to remove any formulation residue.Then removed skin was dipped in hot water (60-80 °C) for 3-5 s, to detached epidermis from dermis.The detached skin layers are further cut in to fragments and mixed with 5 mL ACN and kept for 12 h at 32 ± 1 °C to completely extract ETO.Drug content was evaluated (as discussed earlier) in both epidermis and dermis layers 42,50 .Data obtained from above was fitted in one-compartment model according to equation below Eq. ( 6): In equation, C skin is the drug concentration present in skin at time t, K p is the permeation constant, C Skin max is the maximum drug concentration reached in skin, K e is the skin drug elimination constant.These dermatokinetic parameters K p , C Skin max , T Skin max (time required to achieve C Skin max ) are computed using excel sheet software and for area under the curve (AUC 0-12 h ) 50,51 .

In vitro cell culture analysis
The aim of conducting in vitro cell culture analysis in topical formulations is to assess the potential effects of the formulation on cultured cells, providing insights into its compatibility, safety, and potential efficacy.This analysis helps in understanding cellular responses, absorption rates, and possible cytotoxicity, aiding in the evaluation and refinement of the formulation's characteristics for effective and safe application.

Cell culture and growth conditions
Cell line studies were carried out using human epidermal keratinocyte cell lines (HaCaT) obtained from NCCS, Pune, India.HaCaT cell line were grown in the keratinocyte serum free (KSF) medium and further it was placed in incubator at 37 °C containing 5% CO 2 gas in the environment.The cultured growth cells were again washed with phosphate buffer pH 7.4 solution free from any calcium/magnesium ions.The cell layers were further separated by the addition of 2.0 mL of 0.53 mM EDTA solution with 0.05% w/v of trypsin.The separated cells were diffused smoothly by pipetting into the fresh KSF medium with 10% fetal bovine serum.Further, suspension containing cells was subjected to centrifugation at 125 g and 4 °C for 5 min.The sedimented cells were further suspended into fresh KSF medium at a density of 10,000 cells per well in 96-well plates and 50,000 cells per well in 6-well culture plate and incubated in the CO 2 incubator 42 .www.nature.com/scientificreports/

MTT assay
The investigation focused on the examination of cell proliferation using a modified MTT (3-(4,5,-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay.The HaCaT cells, namely the lx104 cells, were cultured in KFS medium within 96-well plates.The cell suspension was subjected to treatment with a pure drug solution (consisting of a hydro-alcoholic solution) as well as a suspension of DE (made in KSF medium).The control cells were administered exclusively with inert substances, namely a hydro-alcoholic solution and a blank DE suspension.Following the application of the solution treatment, the cells were subjected to incubation within a controlled incubator environment at a temperature of 37 °C for a duration of 48 h.The cells were undergoing washing step using a phosphate buffer.Following that, a measured portion of 150µL of a sterile MTT solution (2.5 mg/mL in phosphate buffer saline) was introduced into every well.The culture plates were subjected to stirring for a duration of 2 h at a temperature of 37 °C using a plate-shaker.Following this, the plates were placed in a CO 2 incubator for incubation.The plates underwent centrifugation at a force of 1200 g the acceleration due to gravity for a duration of 15 min.The liquid portion was discarded, while the MTT formazan crystals were solubilized in 200µL of dimethyl sulfoxide (DMSO).Additionally, a stirring process was conducted for a duration of 20 min, during which the optical density (OD) was assessed at a wavelength of 570 nm 52,53 .

Determination of cellular uptake
For determination of cellular uptake of coumarin-6 dye loaded DE suspension by HaCaT cell lines, lx104 cells were seeded on a 12-well cell culture plate in keratinocyte growth medium (Gibco).The cell culture medium was substituted with mixture of media and the dye loaded DE suspension (20 µg/mL) incubated for 3 h at 37 °C.Before imaging, cells were washed 3 times with 1 mL of PBS.Hereafter, the cells were fixed with 4% paraformaldehyde solution and were viewed under florescence microscope (EVOS) under GFP and blue filter 49 .

Chemical stability
The final formulation is a gel, henceforth only the final formulation was placed for the stability studies.The stability of the developed DE-gel was performed according to ICH guidelines at controlled temperatures i.e., 25 °C ± 2 °C/60% ± 5% RH and at 40 °C ± 2 °C/75% ± 5% RH for 6 months to examine the shelf-life and storage condition of the prepared formulation 54 .

Physical stability
The physical stability of DE-gel was examined in order to study potential changes in organoleptic characteristics after storage.The DE-gel formulations were placed at controlled room temperature of 25 °C ± 2 °C/60% ± 5% RH and at 40 °C ± 2 °C/75% ± 5% RH storage conditions for 6 months to examine the physical change (like discoloration), odour change, crystal formation, consistency of gel, phase separation and change in pH 54 .Confirms that the authors complied with the ARRIVE guidelines.

Skin compliance studies
Female Balb/c mice were used to determine the patient skin-friendly properties of the DE-gel vis-à-vis conventional product using Modified Draize patch test.To avoid any probability of scepticism, female BALB/c mice were categorised into three groups, with 4 animals in respective groups (n = 4).Group A dermal application of normal saline which served as untreated group; Group B dermal application of conventional gel formulation and Group C dermal application of DE-gel formulation.
To initiate the procedure, firstly hairs were plucked off aiding a 0.15 mm animal hair clipper, faced on the skin dorsal side.Around 0.5 g of both the gel formulations were applied uniformly on the clipped off area of mice skin.Furthermore, the tissue was evaluated on the basis of physical examination as well as the histopathological assessment.In the former any visual changes like redness was observed for a period of 7 days after its application measuring the average erythemal grades ranging from 0 to 4 recorded in accordance with the severity of erythema viz.0 = erythema absence, 1 = Slight observation of erythema (light pink), 2 = Moderate erythema (dark pink), 3 = Moderate to severe erythema (light red), 4 = Severe erythema (dark red) 43,55 .

Xylene-induced ear edema model
The activity was carried out on male BALB/c mice to study the effect of xylene-induced ear edema model.The procedure initiates by inducing cutaneous inflammation using xylene in conscious male BALB/c mice via topical route.Prior to the application of the irritant, the gel formulations were administered 30 min in advance during the testing process.The animals were categorized into three discrete groups (n = 4 per group).Group A: dermal application of normal saline (control); Group B: dermal application of conventional gel and Group C dermal application of DE-gel formulation 56,57 .www.nature.com/scientificreports/ The irritant was injected with a micropipette (50 µL/ear) to the interior of the right ear and the left ear represented as control.After 30 min of Xylene application, the mice were immolated by cervical dislocation and the two of the ears (left and right) were expunged instantaneously, by creating parallel cuts throughout the surface of the ear in a hollow pattern.After excision, both the ears were measured and swelling percentage was computed as in Eq. ( 7): where, A as weight of left ear and B as weight of right ear.

Anti-arthritic activity: CFA induced Arthritis in Wistar rats
To assess the anti-arthritic activity, CFA induced arthritis model in female Wistar rats is the generally adopted method.The procedure initiates by division of rats into four groups (n = 4 per group).Group A: dermal application of normal saline (untreated); Group B: served as diseased control.Group C: dermal application of conventional gel and Group D: dermal application of DE-gel formulation.Here the animals were anesthetized using diethyl ether and 100 µl of CFA was injected via into the right hind foot pad Wistar female rat on day 1 intradermally.Hereafter, the animals were observed for arthritis each day.The CFA holds 1 mg of heat killed bacterium in each mL along with 0.85 mL paraffin oil and 0.15 mL mannide mono-oleate.The therapeutic course of medication begins on the 8 th day after the complete introduction of arthritis and further it was sustained for two weeks.Both the novel DE-gel and conventional were applied once daily on knee joints and paws of respective group of animals 56,58 .

% Arthritis swelling reduction
At various time intervals, thickness of paw was measured for each animal 58,59 .Evaluation of each paw for inflammation and arthritis-swelling reduction was measured as given in Eq. ( 8):

Histopathological studies
In order to document any modifications in the skin and paw joint of an arthritic rat model injected with CFA and treated with drug formulations, histopathological investigations were conducted.Following the sacrifice of the animals, skin samples were collected, processed accordingly, and stained with haematoxylin and eosin.The processed skin samples were then examined under a microscope to identify any histopathological alterations 55,60 .

Statistical analysis
A two-way analysis of variance (ANOVA) is used on the data, and then Tukey's multiple 16 comparison test is performed.Statistical significance had been selected to apply when the p < 0.05, unless otherwise noted.

Micromeritics
The size of vesicles is a crucial factor that significantly influences the ability of vesicular systems to penetrate the skin.Fig. 2A  www.nature.com/scientificreports/found to be 102.4± 8.9 nm.The PDI of the vesicles was found to be 0.319 ± 0.016 (less than 1), promising constricted distribution of the polydispersed phase.The zeta potential is a critical factor in assessing the stability of formulations and the capacity of drug delivery systems to adhere to the biological surfaces.The zeta potential of the prepared DE formulation was determined to be 5.05 ± 2.15 mV, as illustrated in Fig. 2B.The present formulation is comprised on non-ionic surfactants and such systems are not stabilized by electrostatic repulsions as in the cases with the dispersion with zetapotential ≥ ± 25 mV.Such systems are also stable owing to steric repulsion.However, the final formulation was a gel, therefore this zetapotential was sufficient enough for the stable nano dispersion gelled in a carbomer-based hydrogel [61][62][63] .

Drug entrapment efficiency and drug content
The percentage drug entrapment from the DE vesicular suspension was found to be 81.87% and the drug content of the DE-gel was found to be 100.04%.This high drug entrapment might be plausible due to the presence of ethanol and cap MCM C10 that would have resulted in better solubilization for the drug and its localization within the interiors of the vesicles.

Vesicle count
To find the population of vesicles in prepared formulation is important parameter for drug entrapment.It was found that the density of un-sonicated DE vesicular population was found to be 242,500 vesicles/mL.The findings confirmed that the sufficient vesicles were formed to enclose the active drug compound.

Deformability index
The deformability index value of DE formulation was found to be 0.91.The optimized DE formulation was found to cross a pore around 2-times smaller to the intact sizes.This property ratified the selection of right edge activator i.e., SDC and ETH in the concentrations to obtain biologically permeable vesicles.The DE of 102.4 nm size has quite sufficient flexibility (i.e., ~ 2 times) to cross the skin barrier pores.The ideal size of the vesicular carriers to cross the skin barriers is around 80-120 nm 64 .Henceforth, the developed system can cross the skin barriers without compromising on shape/losing drug load.Due to inherent deformability of the vesicles, selection can be justified for efficient epicutaneous delivery of ETO.The drug entrapment after extrusion was also determined and the values were non-significantly different than the non-extruded system.

Morphological evaluation
The study employed field emission scanning electron microscopy (FESEM) to investigate the morphology and aggregation of DE, as well as its surface structure and morphological characteristics.The DE suspension was observed using a three-dimensional visualization technique, which revealed that the particles had a spherical shape, demonstrated polydispersity, and did not exhibit any evidence of aggregation, as shown in Fig. 3A (at magnification of 220 ×) and Fig. 3B (at magnification of 20,000 ×).

Calorimetric studies
The fusion characteristics of crystalline materials were examined using the differential scanning calorimetry (DSC).The drug (ETO) displayed a sharpened endothermic peak at a temperature of 153.11 °C, which closely corresponds to the published melting point of the drug (i.e., 145-143 °C).The observed melting behaviour indicates that the drug is in a crystalline state rather than an amorphous state.The other excipients like PL 90G, Cap MCM C10, Cr A25 and SDC exhibited respective endothermic peaks at 62.32 °C, 29.97 °C, 55.84 °C and 74.55 °C corresponding to their reported melting point 40,[65][66][67] .Likewise, the developed DE formulation displayed an endothermic peak at 99.89 °C and the disappearance of drug peak may be ascribed to the encapsulation of drug in the interiors of the developed system.The respective DSC thermograms of are depicted in Fig.  www.nature.com/scientificreports/

Evaluation of ETO loaded-DE gel formulation pH measurement
The pH of developed DE gel was found to be 6.2, which is close to skin i.e., 4.5-6.4 as per literature.As pH of the formulation is neutral or closer to the pH of the skin, the formulation can be considered as safe or may not cause any skin irritation on application.

Rheological studies
The micro-mechanical properties of gels can be assessed by viscometric analysis.The flow properties of DE-gel were recorded by using cone and plate viscometer and the values of parameters were computed using Herschel-Bulkley model.The value of flow index (n = 0.252), obtained from the slope (Fig. 6A), is less than 1 which indicates DE-gel formulation observed a pseudoplastic (shear-thinning) behavior, thus, reflecting decreased formulation viscosity with increase in shear rate (Fig. 6B).The appearance of pseudoplastic behaviour can be attributed to the underlying colloidal network structure, which exhibits deformation and adjustment in response to the direction of flow.The flow behaviour observed in this context might be associated with the existence of microstructures within a three-dimensional lattice network.The consistency index K (Pa.s n ) and yield value were found to be 109.18Pa and 120.55 respectively, which could be due to gel structure rigidity within the hydrogel which required more force to initiate its flow.However, the intrinsic viscosity of DE-gel was found to be 106.7 Pa.s.Attaining a specific phase and viscosity is a crucial prerequisite in the formulation of DE-gel to facilitate their convenient transportation and storage at an optimal temperature.

Texture analysis
Figure 6C and Table 1 both represent the textural curve along with the various values of textural parameters such as firmness, consistency, cohesiveness, and index of viscosity that were acquired from texture analysis of the developed DE-gel formulation.The visual illustration of the DE-gel formulation shows better gel strength, suggesting greater capability to hold the gel at the topical site for a longer period of time, exhibiting smooth extrudability, and ensuring that the gel is easy to spread.www.nature.com/scientificreports/

Ex vivo permeation and drug deposition studies on mouse skin
The final DE-gel was finally studied for desired attributes with regard to permeation of ETO across skin vis-à-vis the conventional gel of equivalent strength.The data presented in Fig. 7 demonstrates the permeation profiles of two formulations.It is evident that the developed DE-gel formulation exhibiting 31.33%drug permeation across of the rodent skin, exhibited superior transport properties compared to the conventional gel formulation (10.34%ETO permeation).This study provides evidence for the better transport properties of the newly developed Table 1.Shows the data for the various parameters obtained from the texture analysis of the developed DE-gel formulation.www.nature.com/scientificreports/carrier-based formulation in comparison to the conventional formulation.The enhanced penetration of ETO from the DE-gel formulation can be attributed to the phospholipid's compatibility with the skin, which is in turn attributable to the effective drug movement properties of the vesicular carriers.Similar findings with phospholipids have been frequently reported and these make the phospholipids are one of the important constituents of the topical formulations 68 .Figure 7 Inset depicts the results of drug deposition in skin reveal that the DE-gel formulation exhibits 648.68 µg/cm 2 (i.e., 33.13%) and conventional gel exhibits 304.08 µg/cm 2 (i.e., 14.71%).DE-gel formulation is 2.25 times significantly higher drug deposition than that of conventional gel (p < 0.01).This suggested DE-gel effectively makes the drug more readily available within the different dermal layers, also depositing them within target sites by means of closely integration of phospholipids to the lipids of the skin.

Skin depth profiling using confocal microscopy
The CLSM technique was used to determine the extent and pattern of DE formulation penetration into the skin layers.The efficacy of a coumarin-6 dye loaded-DE formulation was assessed via topical administration on rat skin for a duration of 6 h.The Fig. 8A depicts the skin treated with coumarin-6 dye alone and Fig. 8B depicts transdermal penetration of coumarin-6 dye loaded-DE formulation across the skin barrier.The confocal images demonstrated that the carrier containing the dye was evenly dispersed throughout the stratum corneum, epidermis, and dermis, exhibiting a significantly higher fluorescent intensity.The findings are consistent with prior literature indicating that the utilization of a flexible vesicular system is significant in enhancing drug solubility and optimizing the distribution of the drug molecule into skin tissue 69 .

Dermal kinetic studies
Dermatokinetic investigations were conducted to assess the dermal pharmacokinetics therapeutic efficacy of the newly formulated DE-gel in comparison to the conventional gel in distinct skin compartments, specifically the epidermis and dermis.Figure 8C and D demonstrate a difference in the permeation of ETO concentration between the epidermal and dermal skin layers following a single application of conventional gel and DE-gel.This observation indicates that the drug concentration over time followed the principles of a one-compartment open body model (1CBM) through the use of dermatokinetic modelling.The administration of DE-gel loaded with ETO exhibited a statistically significant increase (p < 0.05) in the transdermal delivery when compared to the conventional gel.Table 2 displays the values of the following parameters like AUC 0-12 h (µg/ cm 2 ), C Skin max (µg/cm 2 ), T Skin max (h) and K p (h −1 ).It is evocative from the results that the duration of stay of the drug was strongly augmented by the developed DE-gel formulation in deep skin layers and T Skin max (h) decreased markedly.The lower T max by developed   www.nature.com/scientificreports/

MTT assay
The cell viability assay results using MTT dye are depicted in Fig. 9A.HaCaT cells were used to study the cytotoxic possibility of the formulations.The % viability of untreated cells which served as the control was considered to be 100%.The developed formulation i.e., DE and comparative blank DE without ETO did not show any toxicity till 100 µg/mL after 48 h treatment with almost 91.99% and 93.88% viability.In contrast, the pure drug, namely ETO, demonstrated notable toxicity, resulting in less than 21.5% viability in comparison to the control.The findings confirmed that the formulations developed in-house exhibit no cytotoxic effects on HaCaT cells.Although there was no statistically significant variance in the percentage of cell viability comparing the control group and the created formulations, there was a statistically significant variance between the control group and the pure medication (p < 0.05).The observed phenomenon may be attributed to the biocompatible properties inherent in phospholipids.

Determination of cellular uptake
The findings of the investigations on cellular uptake portray the internalization of Coumarin-6 loaded DE formulation by HaCaT cells.As demonstrated in Fig. 9C, the cellular uptake assay of Coumarin-6 DE revealed the proficient internalization of the formulation within the cytosol of HaCaT cells within a time frame of 3 h.The test formulations were observed to induce a distinct green fluorescence in the nuclei of cells, as evidenced by the visualization of coumarin-6.Similarly, coumarin-6 dye labelling alone demonstrated the efficient assimilation of the developed formulations by keratinocytes, as illustrated by the green fluorescence in Fig. 9B.

Chemical stability
Results of stability testing of the developed formulations indicated all the formulations were stable.As discerned from the results given in Fig. 10, the ETO loaded DE-gel formulation is stable at all the studied storage conditions for 6 months.The effect of temperature on the drug migration from one phase to other was not substantial and was suited for topical products.Effect of temperature on the drug content was found to be insignificant at high temperature conditions (40 °C ± 2 °C/75% ± 5% RH) which can be ascribed to the bilayer packing alteration of vesicles at high temperatures.Similarly, the DE-gel formulation stored at controlled room temperature (25 °C ± 2 °C/60% ± 5% RH) conditions also showed better stability for the studied period.

Physical stability
The observations for a period of 6 months were recorded for various physical parameters are enlisted in Table 3.The DE formulation showed macroscopic stability on the studied parameters for 6 months at 25 °C ± 2 °C/60% ± 5% RH and 40 °C ± 2 °C/75% ± 5% RH storage conditions.The DE-gel formulation was devoid of notable discoloration and change in odour.The gel consistency also remained good with absence of drug crystals and phase separation.The particle size alteration was below 13%, indicating acceptable variation.However, the present study is limited in scope w.r.t. the changes in the lamellae and viscosity.www.nature.com/scientificreports/

Skin compliance studies
The developed DE-gel formulation was evaluated for any irritating effect on the skin.The erythemal grading (ranging from 0 to 4) were recorded for 7 days.Absence of erythema on skin was observed in case of DE-gel formulation, whereas moderate to severe erythema (light red) scores were observed in case of conventional product as shown in Fig. 11 and the scoring is tabulated in Table 4.
In conformity with the histopathology, the skin section of various animal groups treated with DE-gel formulation and conventional gel formulation were stained with eosin-hematoxylin and evaluated for the histological changes occurred during the period of exposure.Figure 11A showed the photograph of untreated skin, which was normal.Figure 11B showed epidermal thickening and inflammation in the dermis layer on the skin treated with conventional gel.Therefore, the microscopic examination directed that the viable formulation had compromised the normal healthy skin.Furthermore, the skin section treated with DE-gel formulation was found to be healthy with no inflammation in the dermis tissue.It revealed DE-gel did not damage the normal healthy skin Fig. 11C.The study's findings demonstrated the safety and effectiveness of biocompatible phospholipid in the DE-gel system.These positive outcomes can be due to phospholipids interaction with skin components and their ability to establish a skin-depot.The study reported here align with prior literature, which suggests that lipid-based formulations are both safer and more compatible with the skin 55,56 .

Anti-inflammatory assessment
Xylene-induced ear edema model.Figure 12A showed DE-gel formulation exhibited remarkably advanced anti-inflammatory activity versus conventional formulation.The % swelling of treated ear was reduced by 2.99 times (DE-gel formulation) and 1.33 times (conventional gel formulation) with respect to untreated ear.Thus, the efficacy of the formulated DE-gel was significantly 2.2-folds higher than that of conventional (p < 0.01).The outcomes of the animal study conducted exposed the edge of the vesicular delivery systems as compared to the conventional systems.This accredited to their better interaction with the skin and skin-depot forming potential.As per the histopathological studies, there was division into three groups viz.disease control causing swelling, epidermis stretching and detachment of epidermis from dermis as seen in Fig. 12B.The histopathology of untreated ear as shown in Fig. 12C was normal whereas Fig. 12E displayed DE-gel group with healing of ear with intact epidermal and dermal layers and no edema formation.In contrast, Fig. 12D was the group treated with conventional gel formulation reporting incomplete recovery which was manifested from histopathology where disordered articular cartilage with greater number of inflammatory cells was experienced.
Anti-arthritic activity: CFA induced arthritis in wistar rats.To understand the anti-arthritic activity in diseased rats after topical application of DE-gel formulation, the most vital parameter that is % arthritis swelling reduction was calculated.A significant rise in swelling was measured for CFA rats which didn't receive any treatment.In the conventional gel, a slight reduction in % swelling was detected (i.e., 27.27%), however in the DE-gel formulation, a significant decrease in swelling was noted (i.e., 3.89%), as depicted in Fig. 13A.Thus, efficacy of the DE-gel formulation was sevenfold higher than that of group treated with conventional gel formulation (p < 0.01).This shows the superior activity of ETO loaded DE-gel over conventional gel in arthritis and indicated better penetration of drug to the site of action.
The histological examination of paw joint and paw skin of animals suffering from arthritis treated with different ETO formulations was executed to evaluate the level of inflammation and morphological behaviour in the internal structure of the paw joint and paw skin.
Figure 13C represents paw joint of CFA (untreated) induced arthritic rat experienced accretion of synovial fluid, lymphocytes in synovium, tissues granulation along with formation of pannus adding up to the high level of inflammation extended into joint synovium recorded as compared to the normal joint (Fig. 13B).Figure 13D presents a little lower inflammation with the joint space surrounded by inflammatory cells with moderate pannus formation and accretion of synovial fluid in the case of paw joints treated with conventional gel formulation.Almost no signs of inflammation were observed in the paw joint treated with DE-gel formulation attaining improved joint bone health to its normal structure as seen in Fig. 13E.
Similar results were recorded for the infected paw skin in Fig. 14 further divided into 4 groups i.e., control, untreated, conventional gel, DE-gel treated rat. Figure 14B showed acute inflammation, augmented thickness of the layers, disrupted layers with separation and hyperkeratosis in the untreated rat paw skin.The normal paw skin was observed to be intact having the natural anatomy of skin (Fig. 14A).In comparison to the rat paw skin  treated with conventional gel which showed moderate inflammation with slightly higher thickness of the skin layers, edema and hyperkeratosis in Fig. 14C vis-à-vis negligible level of inflammation was detected in case of DE-gel treated rat skin as shown in Fig. 14D.The severity degree for treated groups is described as a score of inflammation and arthritis from 0 to + + + + (0 indicates normal & + + + + indicates severe).The severity of inflammations for paw skin was observed in the following order: CFA (untreated > conventional gel formulation > DE-gel formulation = control (normal rat).

Conclusions
The current research endeavor was undertaken to enhance the topical administration attributes of etodolac by investigating the potential of deformable-emulsomes formulated with sodium deoxycholate (SDC).The selection of SDC as the novel excipient was based on its biocompatibility and the flexibility enhancement potential that can translate into enhanced permeability of bioactive substances across the skin.The system that was developed exhibited superior characteristics in terms of ex vivo permeation profile, drug retention, and in vivo anti-inflammatory activity.This can be attributed to the enhanced deformability of the developed vesicles compared to the conventional formulations.As a result, these vesicles were able to effectively penetrate deeper layers of the skin.FESEM, CLSM and dermatokinetic investigations revealed that the flexible vesicles generated were spherical, homogenous and fairly dispersed the drug across the subcutaneous, epidermis, and dermis.
In conclusion, the current research confirmed that the formulation of deformable emulsomes alone and it's gel both can effectively promote the localization of the drug at the site of action in the desired amounts.The methodology that has been implemented has facilitated the development of an effective and regulatory compliant formulation of etodolac for topical usage.The outcomes from this investigation are highly promising and indicate that these biocompatible carriers have the potential to be utilized as a therapeutic approach for managing

FormulationFigure 7 .
Figure 7. Ex vivo permeation profile of DE-gel and conventional gel formulations.Each bar indicates ± SD (n = 3).Inset figure depicting bar diagram for the amount of drug deposited by the skin for DE-gel and conventional formulations.Each cross bar indicates ± SD. (n = 3).

Figure 8 .
Figure 8. (A) CLSM of coumarin-6 dye alone; (B) coumarin-6 dye loaded-DE formulation in the deeper layer of the skin; (C) ETO concentration time profile in epidermis and dermis of Wistar rats after single application of Conventional gel formulation; (D) ETO concentration time profile in epidermis and dermis of Wistar rats after single application of DE-gel formulation.Each cross bar indicates ± SD (n = 4).

Figure 9 .
Figure 9. (A) Cell viability assay results after 48 h of treatment of pure drug ETO, ETO-DE, blank DE.The results are expressed as % cell viability, taking the viability of control as 100%.Each cross bar indicates ± SD (n = 4;****p < 0.0001; ns-non significant); (B) Images depict HaCaT cells subjected to treatment with coumarin-6 alone at 40 ×; and (C) Coumarin-6 loaded DE at 40 × objective, respectively, as observed through fluorescent microscopy.

Figure 10 .
Figure 10. Figure depicting the % drug assay of DE-gel at different storage conditions and time intervals.

Figure 13 .
Figure 13.(A) Comparison of % arthritis swelling after application of DE-gel formulation and conventional gel formulations.Each cross bar indicates ± SD (n = 4); (B) Histopathology of normal paw joint; (C) Disease control paw joint; (D) conventional gel formulation treated paw joint; (E) DE-gel formulation treated paw joint.

Table 2 .
Various dermatokinetic parameters (Mean ± SD) of ETO topical formulations in epidermis and dermis.